Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) activity is frequently deregulated in cancers of many etiologies. The mechanisms governing its activity in normal and pathological settings are incompletely understood, particularly with respect to amino acid sensing, an important contributor to its activation. Characterization of the processes controlling mTORC1 activation by amino acids may identify novel targets with broad application to cancer treatment.

Amino acid sensing requires the Vacuolar H+-ATPase (V-ATPase), an ATP-dependent proton pump primarily responsible for the pH control of intracellular compartments. While it is known that the intact V-ATPase is necessary for mTORC1 activation by amino acids, the mechanism of its involvement unknown. We thus sought to determine if amino acids affect activity of the V-ATPase by assessing V-ATPase-dependent fluorescence quenching in lysosomes loaded with FITC-Dextran (a pH sensitive dye). Our data demonstrate that amino acid starvation increases V-ATPase activity, and that readdition of amino acids reverses this effect. We then demonstrated that amino acid starvation increases V-ATPase assembly, a main mode of regulation of the enzyme whereby association of the two functional domains, V1 and V0, is altered to increase or decrease the amount of active V-ATPase. Similarly to the change observed in activity, the change in V-ATPase assembly is reversed by amino acid readdition. These changes do not depend on PI3K or mTORC1 activity, both of which have been linked to changes in V-ATPase activity and assembly in response to other stimuli, such as glucose and growth factor stimulation. Further, we sought to determine if specific amino acids recapitulate the effect of total amino acid starvation, and preliminary data suggest that leucine and lysine both play important roles in controlling V-ATPase activity. These studies demonstrate that amino acid sensing controls V-ATPase activity and assembly, which may be important in subsequent signaling to mTORC1.